Photoperiodic regulation of the orexigenic effects of ghrelin in Siberian hamsters

Animals living in temperate climates with predictable seasonal changes in food availability may use seasonal information to engage different metabolic strategies. Siberian hamsters decrease costs of thermoregulation during winter by reducing food intake and body mass in response to decreasing or short-day lengths (SD). These experiments examined whether SD reduction in food intake in hamsters is driven, at least in part, by altered behavioral responses to ghrelin, a gut-derived orexigenic peptide which induces food intake via NPY-dependent mechanisms. Relative to hamsters housed in long-day (LD) photoperiods, SD hamsters consumed less food in response to i.p. treatment with ghrelin across a range of doses from 0.03 to 3 mg/kg. To determine whether changes in photoperiod alter behavioral responses to ghrelin-induced activation of NPY neurons, c-Fos and NPY expression were quantified in the arcuate nucleus (ARC) via double-label fluorescent immunocytochemistry following i.p. treatment with 0.3 mg/kg ghrelin or saline. Ghrelin induced c-Fos immunoreactivity (-ir) in a greater proportion of NPY-ir neurons of LD relative to SD hamsters. In addition, following ghrelin treatment, a greater proportion of ARC c-Fos-ir neurons were identifiable as NPY-ir in LD relative to SD hamsters. Changes in day length markedly alter the behavioral response to ghrelin. The data also identify photoperiod-induced changes in the ability of ghrelin to activate ARC NPY neurons as a possible mechanism by which changes in day length alter food intake.     

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.     

Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.     

CCK inhibits the orexigenic effect of peripheral ghrelin

CCK and ghrelin exert antagonistic effects on ingestive behavior. The aim of the present study was to investigate the interaction between ghrelin and CCK administered peripherally on food intake and neuronal activity in specific hypothalamic and brain stem nuclei, as assessed by c-Fos-like immunoreactivity (c-FLI) in nonfasted rats. Ghrelin (13 microg/kg body wt) injected intraperitoneally significantly increased the cumulative food intake when measured at 30 min and 1 h after injection, compared with the vehicle group (2.9 +/- 1.0 g/kg body wt vs. 1.2 +/- 0.5 g/kg body wt, P < 0.028). Sulfated CCK octapeptide (CCK-8S) (2 or 25 microg/kg body wt) injected simultaneously blocked the orexigenic effect of ghrelin (0.22 +/- 0.13 g/kg body wt, P < 0.001 and 0.33 +/- 0.23 g/kg body wt, P < 0.0008), while injected alone, both doses of CCK-8S exerted a nonsignificant trend to reduce food intake. Ghrelin (13 microg/kg body wt ip) markedly increased the number of c-FLI-positive neurons per section in the arcuate nucleus (ARC) compared with vehicle (median: 31.35 vs. 9.86, P < 0.0001). CCK-8S (2 or 25 microg/kg body wt ip) had no effect on neuronal activity in the ARC, as assessed by c-FLI (median: 5.33 and 11.21 cells per section), but blocked the ghrelin-induced increase of c-fos expression in this area when both peptides were administered simultaneously (median: 13.33 and 12.86 cells per section, respectively). Ghrelin at this dose had no effect on CCK-induced stimulation of c-fos expression in the paraventricular nucleus of the hypothalamus and the nucleus of the solitary tract. These results suggest that CCK abolishes ghrelin-induced food intake through dampening increased ARC neuronal activity.     

Central mechanisms involved in the orexigenic actions of ghrelin

Ghrelin is a stomach hormone, secreted into the bloodstream, that initiates food intake by activating NPY/AgRP neurons in the hypothalamic acruate nucleus. This review focuses on recent evidence that details the mechanisms through which ghrelin activate receptors on NPY neurons and downstream signaling within NPY neurons. The downstream signaling involves a novel CaMKK-AMPK-CPT1-UCP2 pathway that enhances mitochondrial efficiency and buffers reactive oxygen species in order to maintain an appropriate firing response in NPY. Recent evidence that shows metabolic status affects ghrelin signaling in NPY is also described. In particular, ghrelin does not activate NPY neurons in diet-induced obese mice and ghrelin does not increase food intake. The potential mechanisms and implications of ghrelin resistance are discussed.     

Peripheral ghrelin stimulates feeding behavior and positive energy balance in a sciurid hibernator

Hibernators exhibit a robust circannual cycle of body mass gain and loss primarily mediated by food intake, but the pathways controlling food intake in these animals have not been fully elucidated. Ghrelin is an orexigenic hormone that increases feeding in all mammals studied so far, but has not until recently been studied in hibernators. In other mammals, ghrelin stimulates feeding through phosphorylation and activation of AMP-activated protein kinase (AMPK). Activation of AMPK phosphorylates and deactivates acetyl Co-A carboxylase (ACC), a committed step in fatty acid synthesis. In order to determine the effects of exogenous ghrelin on food intake and metabolic factors (i.e. non-esterified fatty acids (NEFAs), and hypothalamic AMPK and ACC) in hibernators, ghrelin was peripherally injected into ground squirrels in all four seasons. Changes in food intake and body mass were recorded over a 2-6 hour period post injections, and squirrels were euthanized. Brains and blood were removed, and Western blots were performed to determine changes in phosphorylation of hypothalamic AMPK and ACC. A colorimetric assay was used to determine changes in concentration of serum NEFAs. We found that food intake, body mass, and locomotor activity significantly increased with ghrelin injections versus saline-injected controls, even in animals injected during their aphagic winter season. Injected ghrelin was correlated with increased phosphorylation of AMPK, but didn't have an effect on ACC in winter. Ghrelin-injected animals also had increased levels of serum NEFAs compared with saline controls. This study is the first to show an effect of injected ghrelin on a hibernator.     

The octanoylated energy regulating hormone ghrelin: An expanded view of ghrelin’s biological interactions and avenues for controlling ghrelin signaling

Ghrelin is a small peptide hormone that requires a unique post-translational modification, serine octanoylation, to bind and activate the GHS-R1a receptor. Initially demonstrated to stimulate hunger and appetite, ghrelin-dependent signaling is implicated in a variety of neurological and physiological processes influencing diseases such as diabetes, obesity, and Prader-Willi syndrome. In addition to its cognate receptor, recent studies have revealed ghrelin interacts with a range of binding partners within the bloodstream. Defining the scope of ghrelin’s interactions within the body, understanding how these interactions work in concert to modulate ghrelin signaling, and developing molecular tools for controlling ghrelin signaling are essential for exploiting ghrelin for therapeutic effect. In this review, we discuss recent findings regarding the biological effects of ghrelin signaling, outline binding partners that control ghrelin trafficking and stability in circulation, and summarize the current landscape of inhibitors targeting ghrelin octanoylation.     

Reduced intake of carbohydrate prevents the development of obesity and impaired glucose metabolism in ghrelin O-acyltransferase knockout mice

A close relationship between acylated-ghrelin and sucrose intake has been reported. However, little has been examined about the physiological action of ghrelin on preference for different types of carbohydrate such as glucose, fructose, and starch. The current study was aimed to investigate the role of acylated-ghrelin in the determinants of the choice of carbohydrates, and pathogenesis of chronic disorders, including obesity and insulin resistance. In a two-bottle-drinking test, ghrelin O-acyltransferase (GOAT) knockout (KO) mice consumed a less amount of glucose and maltodextrin, and almost the same amount of fructose and saccharin solution compared to WT littermates. The increased consumption of glucose and maltodextrin was observed when acylated-ghrelin, but not unacylated-ghrelin, was exogeneously administered in normal C57BL/6J mice, suggesting an association of acylated-ghrelin with glucose-containing carbohydrate intake. When fed a diet rich in maltodextrin, starch and fat for 12 weeks, GOAT KO mice showed less food intake and weight gain, as well as improved glucose tolerance and insulin sensitivity than WT mice. Our data suggests that blockade of GOAT activity may offer a therapeutic option for treatment of obesity and its associated metabolic syndrome by preventing from overconsumption of carbohydrate-rich food.     

Motivation to obtain preferred foods is enhanced by ghrelin in the ventral tegmental area

Ghrelin is an orexigenic peptide that acts within the central nervous system to stimulate appetite and food intake via the growth hormone secretagogue receptor (GHS-R). It has been hypothesized that ghrelin modulates food intake in part by stimulating reward pathways in the brain and potentially stimulating the intake of palatable foods. Here we examined the effects of chronic ghrelin administration in the ventral tegmental area (VTA) via osmotic minipumps on 1) ad libitum food intake and bodyweight; 2) macronutrient preference; and 3) motivation to obtain chocolate pellets. In the first study rats receiving ghrelin into the VTA showed a dose-dependent increase in the intake of regular chow, also resulting in increased body weight gain. A second study revealed that intra-VTA delivery of the ghrelin receptor antagonist [Lys-3]-GHRP-6 selectively reduced caloric intake of high-fat chow and reduced body weight gain relative to control and ghrelin treated rats. The third study demonstrated that food restricted rats worked harder for food pellets when infused with ghrelin than when infused with vehicle or ghrelin receptor antagonist treated rats. Finally, rats trained on an FR1 schedule but returned to ad libitum during ghrelin infusion, responded at 86% of baseline levels when they were not hungry, whereas saline infused rats responded at 36% of baseline. Together, these results suggest that ghrelin acts directly on the VTA to increase preference for and motivation to obtain highly-palatable food.     

Ghrelin, des-acyl ghrelin and obestatin: three pieces of the same puzzle

The major active product of ghrelin gene is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, called simply ghrelin. Ghrelin has a multiplicity of physiological functions, affecting GH release, food intake, energy and glucose homeostasis, gastrointestinal, cardiovascular, pulmonary and immune function, cell proliferation and differentiation and bone physiology. Nevertheless, recent developments have shown that ghrelin gene can generate various bioactive molecules besides ghrelin, mainly des-acyl ghrelin and obestatin, obtained from alternative splicing or from extensive post-translational modification. Although their receptors have not yet been identified, they have already proven to be active, having intriguingly subtle but opposite physiological actions to ghrelin. This suggests the existence of a novel endocrine system with multiple effector elements which not only may have opposite actions but may regulate the action of each other. In this review, we summarize the steps which lead to the production of the different ghrelin gene products and examine the most significant differences between them in terms of structure and actions.     

Interleukin-1 beta-induced anorexia is reversed by ghrelin

Interleukins, in particular interleukin-1β (IL-1β), reduce food intake after peripheral and central administration, which suggests that they contribute to anorexia during various infectious, neoplastic, and autoimmune diseases. On the other hand, ghrelin stimulates food intake by acting on the central nervous system (CNS) and is considered an important regulator of food intake in both rodents and humans. In the present study, we investigated if ghrelin could reverse IL-1β-induced anorexia. Intracerebroventricular (i.c.v.) injection of 15, 30 or 45 ng/μl of IL-1β caused significant suppression of food intake in 20 h fasting animals. This effect lasted for a 24 h period. Ghrelin (0.15 nmol or 1.5 nmol/μl) produced a significant increase in cumulative food intake in normally fed animals. However, it did not alter food intake in 20 h fasting animals. Central administration of ghrelin reduced the anorexic effect of IL-1β (15 ng/μl). The effect was observed 30 min after injection and lasted for the next 24 h. This study provides evidence that ghrelin is an orexigenic peptide capable of antagonizing IL-1β-induced anorexia.     

Effects of ghrelin and des-acyl ghrelin on neurogenesis of the rat fetal spinal cord

Expressions of the growth hormone secretagogue receptor (GHS-R) mRNA and its protein were confirmed in rat fetal spinal cord tissues by RT-PCR and immunohistochemistry. In vitro, over 3 nM ghrelin and des-acyl ghrelin induced significant proliferation of primary cultured cells from the fetal spinal cord. The proliferating cells were then double-stained using antibodies against the neuronal precursor marker, nestin, and the cell proliferation marker, 5-bromo-2'-deoxyuridine (BrdU), and the nestin-positive cells were also found to be co-stained with antibody against GHS-R. Furthermore, binding studies using [125I]des-acyl ghrelin indicated the presence of a specific binding site for des-acyl ghrelin, and confirmed that the binding was displaced with unlabeled des-acyl ghrelin or ghrelin. These results indicate that ghrelin and des-acyl ghrelin induce proliferation of neuronal precursor cells that is both dependent and independent of GHS-R, suggesting that both ghrelin and des-acyl ghrelin are involved in neurogenesis of the fetal spinal cord.     

Ghrelin, des-acyl ghrelin and obestatin on the gastrointestinal motility

Ghrelin, des-acyl ghrelin and obestatin are derived from a common prohormone, preproghrelin by posttranslational processing, originating from endocrine cells in the stomach. Ghrelin exerts stimulatory effects on the motility of antrum and duodenum in both fed and fasted state of animals. On the other hand, des-acyl ghrelin exerts inhibitory effects on the motility of antrum but not on the motility of duodenum in the fasted state of animals. Obestatin exerts inhibitory effects on the motility of antrum and duodenum in the fed state but not in the fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of ghrelin, CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, whereas CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin.     

Gender-specific orexigenic and anorexigenic mechanisms in rats

Feeding dysregulation may manifest as either under-nourishment (e.g., anorexia) or excessive eating leading to obesity. Recent studies have suggested a gender-related variance in weight maintenance in response to chronic disease or obesity-related dietary regimens. However it is unclear whether these gender differences in weight management are secondary to appetite-mediated food intake or alternative mechanisms (e.g., exercise, metabolism). In this study, we explored gender-dependent feeding and hormonal responses to dietary restriction (12-h fast) or to an inflammatory stimulus (LPS, 100 microg/kg b.w.; i.p.) in rats. In response to a 12 h fast, female rats increased (p<0.05) total daily food intake above that of male rats by primarily increasing nighttime feeding by 40%, as compared to 10% in males. Consistent with the increased food intake, fasting induced a greater percent increase in female as compared to male plasma ghrelin (141 vs. 65%, p<0.001). In response to LPS, both male and female rats showed similar reductions in total daily food consumption. However LPS (6 h) induced a greater percent increase in plasma leptin in female than male rats (230 vs. 33%, p<0.01), whereas ghrelin was similarly decreased in both females and males (66 vs. 44%). These findings demonstrate sexual dimorphic responses in feeding and appetite-associated hormonal responses to fasting or LPS treatment. Our findings suggest that therapeutic interventions with ghrelin or leptin must be modified according to gender in order to optimally achieve either weight loss for obesity or weight gain/maintenance for chronic illness-associated anorexia.     

Morphometrical and intracellular changes in rat ovaries following chronic administration of ghrelin

The aim of our investigation was to examine the influence of chronic administration of ghrelin on the rat ovarian state. Morphometrical and intracellular changes in the ovary of 35-d female Wistar rats after sc injection of 1 nmol of ghrelin for 10 consecutive days were studied. Control animals (n = 10) were injected with normal saline using similar method. The ovaries were collected on days 1 and 6 after last injection from each group and subjected to light microscopic morphometric and electron microscopic analysis. It was demonstrated that the number of corpora lutea was significantly lower and the number of ovarian follicles was higher in the treated group on days 1 and 6, than in control (P < 0.01). Moreover, the mean diameter of each follicle, corpora lutea, luteal cell, theca layer, oocyte and zona plucida, but not of granulosa layer, as well as the whole ovarian volume were significantly lower in the treated animals at days 1 and 6 (P < 0.05). Electron microscopic analysis also indicated some intracellular changes associated with apoptosis and cell death such as presence of secondary lysosome, apoptotic bodies, nuclear chromatin condensation as well as margination, nuclear segmentation and vacuolization of cytoplasm of granulosa and theca cells. Our observations provides novel evidences for inhibitory influence of ghrelin on rat ovarian structures and, therefore, for the role of ghrelin as suppressor of female reproductive system.     

Immunohistochemical localization of orexin-B, orexin-1 receptor, ghrelin, GHS-R in the lacrimal gland of normal and diabetic rats

Orexin-B, ghrelin and their receptors play an important role in the regulation of feeding in mammals. The pattern of distribution of orexin-B, orexin-1-receptor (OX₁R), ghrelin and growth hormone secretagogue receptor (GHS-R) in the lacrimal gland of normal and diabetic rats has not been reported. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) at 60 mg kg⁻¹. Forty weeks after the induction of STZ-induced diabetes, normal, age-matched controls and diabetic rats were anesthetized with chloral hydrate (intraperitoneally) and their lacrimal glands removed and processed for immunofluorescence. Orexin-B was observed in the cells localized to the interacinar regions while OX₁R was discerned in the nerves innervating the wall of small blood vessels. Ghrelin was also present in a group of cells located in the periacinar regions of the lacrimal glands of normal and diabetic rats. In contrast, GHS-R was observed in the apical region of the ductal cells of the lacrimal glands of both normal and diabetic rats. The pattern of distribution of these orexigenic peptides and their receptors did not significantly change after the onset of diabetes. In conclusion, orexin-B, ghrelin and their receptors are present in the lacrimal glands of both normal and diabetic rats and may play a role in the regulation of lacrimal gland function.     

下丘脑室旁核orexin-A对大鼠摄食和胃动力影响及调控机制

目的:探讨下丘脑室旁核orexin-A对大鼠摄食和胃动力影响及调控机制。方法:采用免疫组化观察下丘脑室旁核(paraventricular nucleus,PVN)orexin受体表达情况;PVN注射orexin-A观察大鼠摄食、胃运动、胃酸分泌和胃排空的改变。结果:免疫组化实验显示大鼠PVN中存在orexin受体免疫阳性细胞。PVN注射orexin-A后,大鼠前三小时摄食增加,6 h和24 h摄食无显著改变。PVN微量注射orexin-A后,大鼠胃运动幅度和频率增加、胃排空增快并且胃酸分泌增多。[D-Lys-3]-GHRP-6可部分阻断orexin-A对摄食、胃运动、胃排空和胃酸分泌的促进作用,SB334867可完全阻断orexin-A对胃运动、胃排空和胃酸分泌的促进作用。结论:下丘脑室旁核orexin-A可能通过生长激素促泌素GHSR受体信号通路调控大鼠摄食及胃功能。     

The role of ghrelin on the morphometry and intracellular changes in the rat testis

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Expression of the functional ghrelin receptor, GHS-R1a, has been shown in Sertoli and Leydig cells as well as seminiferous tubules. Therefore, we investigated the effects of chronic administration of ghrelin on morphometry of testicular cells and its probable intracellular alterations. Thirty 45-day male Wistar rats were scheduled for the study and were divided into control and treatment groups. In the treatment group, 1nmol of ghrelin was administered as sc injection for 10 consecutive days or vehicle (physiological saline) to the control rats. Testes were taken by killing of rats on days 5, 15 and 40 after last injection and underwent for photomicrograph and electronmicrograph evaluations as well as stereological estimations. Testicular histomorphometry revealed a significant decrease in the different cell types except for spermatogonia in the treatment animals (P<0.01). Such a cellular decrease was also found in the stereological estimations in this group. Likewise, seminiferous tubules diameter and their germinal epithelium thickness decreased in the treated rats (P<0.01). In intracellular observations, much vacuolated mitochondria, limited endoplasmic reticulum, lesser intracellular organels and several detachment areas between cell membrane and its basement membrane were detected in the ghrelin-treated group. These findings indicate that ghrelin has anti-proliferative effects on different testicular cell types and is a negative modulator of male reproductive system.     

Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin

Ghrelin is a peptide hormone produced and secreted from the stomach. Hypothalamic injection of the peptide increases food intake but it is not known if the peptide affects other brain regions. We measured several behavioral parameters such as anxiety (elevated plus maze), memory retention (step down test), and food intake after injections of different doses of the peptide in the hippocampus, amygdala, and dorsal raphe nucleus (DRN). The injection of ghrelin in the hippocampus and DRN significantly and dose dependently increased food intake in relation to controls rats, while injections into the amygdala did not affect the food intake. We also show for the first time that ghrelin clearly and dose dependently increases memory retention in the hippocampus, amygdala, and DRN. Moreover, ghrelin at different potencies induced anxiogenesis in these brain structures while the highest dose of 3 nmol/microl was effective in all of them. The comparison of sensitivity of each brain structure indicates a specific role of them for each of the behaviors studied. The results provide new insight in to the anatomical substrate and the functional role of extrahypothalamic ghrelin targets in the CNS.     

The effect of ghrelin on MK-801 induced memory impairment in rats

Accumulating evidence indicates that the brain-gut peptide ghrelin which is expressed in hippocampus improves memory and learning processes. The MK-801, a noncompetitive NMDA receptor antagonist, has also shown amnesic properties in animal model. The current study was to find out whether intracerebroventricular administration of ghrelin can prevent amnesia induced by MK-801 in rats. A week after the surgery, during which cannuals were implanted in the lateral ventricular, the animals were trained and tested in a step-through type passive avoidance task. Memory retrieval was measured by step-through latency (STL) and total time in dark compartments (TDC). In the first series of experiments, we established a dose–response relationship for ghrelin on the passive avoidance paradigm. In the second set of experiments, animals were divided to two groups. In the first group, MK-801 (0.075, 0.15 and 0.3 mg/kg) was injected intraperitoneally (i.p.) immediately after the acquisition session and in the second group MK-801 (same doses) was injected (i.p.) 30 min before the retention session. Analysis of data showed that in both groups, MK-801 impaired learning and memory. In the third set of experiments, administration of ghrelin (200 ng/rat) right after the acquisition session (i.e. before MK-801 injection) improved the MK-801 induced memory impairment, but administration of ghrelin before retrieval session did not affect the MK-801 induced memory impairment.These results show an interaction between ghrelin and glutamatergic system. A novel finding in this study is that ghrelin can prevent amnesia produced by NMDA antagonist in rats when injected in post-training phase.